WO2022239724A1 - Rhabdovirus modifié ayant une toxicité réduite - Google Patents

Rhabdovirus modifié ayant une toxicité réduite Download PDF

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WO2022239724A1
WO2022239724A1 PCT/JP2022/019633 JP2022019633W WO2022239724A1 WO 2022239724 A1 WO2022239724 A1 WO 2022239724A1 JP 2022019633 W JP2022019633 W JP 2022019633W WO 2022239724 A1 WO2022239724 A1 WO 2022239724A1
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protein
rna genome
cells
vsv
gene encoding
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Yohei Yokobayashi
Narae Kim
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Okinawa Institute Of Science And Technology School Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to an engineered rhabdovirus that may have a reduced toxicity and/or induce a higher replication capability and/or express a gene loaded onto the virus in an infected cell, preferably in an undifferentiated cell.
  • VSV vesicular stomatitis virus
  • VSV vector is an RNA viral vector. It does not have any DNA intermediates, thus no possibility of genomic integration. Genomic integration of a vector sometimes destroys the important part of the host genome. Thus, no possibility of genome integration is a preferable feature in vectors. VSV infection causes mild or no symptoms in humans. Thus, VSV vectors are being developed for applications as oncolytic vectors and vaccine vectors.
  • VSV shows a strong cytotoxicity. This feature of VSV vectors may cause a limitation to their use in these cells.
  • the invention provides an engineered rhabdovirus that may have a reduced toxicity and/or induce a higher replication capability and/or express a gene loaded onto the virus in an infected cell.
  • the inventors found that two newly created mutations synergistically reduce a cytopathogenicity of VSV vectors in mouse embryonic stem cells (ESCs), and thus, can induce an increase in the number or ratio of cells that maintain VSVs and provide a higher expression of a gene carried by the VSVs.
  • ESCs mouse embryonic stem cells
  • the disclosure may provide the invention as follows.
  • An RNA genome of a rhabdovirus comprising at least a gene encoding M protein, wherein the M protein has mutations in methionine residues corresponding to the methionine residues positioned at positions 33 and/or 51 of M protein set forth in SEQ ID No: 1, thereby the rhabdovirus can produce a longer isoform (Isoform M1) whose N terminal amino acid is methionine at position 1 and cannot produce at least one of two shorter isoforms (Isoforms M2 and M3) whose N terminal amino acid is methionine residues corresponding to the methionine residues positioned at positions 33 and/or 51, respectively, wherein the RNA genome lacks gene encoding functional G protein.
  • RNA genome according to (1) above wherein the RNA genome has the gene encoding M protein and a transcription end signal (TES) and the TES has an insertion of an RNA residue inside the TES.
  • TES transcription end signal
  • the RNA residue comprises an adenine ribonucleotide.
  • the RNA genome further comprises at least a gene encoding L protein, and L protein has a mutation in isoleucine corresponding to the isoleucine positioned at 762 of L protein set forth in SEQ ID No. 2.
  • RNA genome according to (2) or (3) above wherein the RNA genome further comprises at least a gene encoding L protein, and L protein has a mutation in isoleucine corresponding to the isoleucine positioned at 762 of L protein set forth in SEQ ID No. 2.
  • RNA genome according to any one of (1) to (5) above wherein the RNA genome further comprises at least a gene encoding L protein, and L protein has a mutation in one or more selected from the group consisting of isoleucine, glutamine, and threonine corresponding to the isoleucine positioned at 762, 1530, and 1910, respectively, of L protein set forth in SEQ ID No. 2.
  • RNA genome according to any one of (1) to (6) above wherein the RNA genome further comprises a gene encoding N protein, and a gene encoding P protein.
  • RNA genome according to any one of (1) to (7) above wherein the RNA genome further comprises at least a gene encoding N protein and the N protein has a mutation in lysin corresponding to the lysin positioned at 155 of N protein set forth in SEQ ID No. 16.
  • the rhabdovirus is a vesicular stomatitis virus (VSV).
  • VSV vesicular stomatitis virus
  • RNA genome according to any one of (1) to (9) above wherein the RNA genome comprises a gene encoding L protein that is operably linked to a guanine-responsive translational switch.
  • the RNA genome according to any one of (1) to (10) above further comprises an expression cassette comprising a gene of interest to be expressed that is operably linked to a control sequence.
  • the control sequence is a guanine-responsive translational switch.
  • a rhabdovirus comprising a virion having the RNA genome according to any one of (1) to (12) above.
  • An expression vector comprising the DNA according to (15) above that is operably linked to a control sequence (e.g., a promoter).
  • a cell preferably, an animal cell, for example, a human cell, preferably, a human pluripotent cell, more preferably a human pluripotent stem cell, or a cell differentiated from the cultured human pluripotent stem cell
  • the disclosure may provide the invention as follows.
  • (18) The RNA genome according to any one of (1) to (12) above, wherein the translational end signal (TES) located at 3’ untranslated region (3’ UTR) has a mutation.
  • (19) The RNA genome according to any one of (1) to (12) above, wherein the translational end signal (TES) located at 3’ untranslated region (3’ UTR) has an insertion with one or more nucleic acid.
  • (20) The RNA genome according to any one of (1) to (12) above, wherein the translational end signal (TES) located at 3’ untranslated region (3’ UTR) has an insertion in the nucleic acid corresponding to the nucleic acid sequence set forth in SEQ ID No. 4.
  • TES translational end signal
  • TES translational end signal located at 3’ untranslated region
  • TES translational end signal
  • TES translational end signal located at 3’ untranslated region
  • a cell preferably, an animal cell, for example, a human cell, preferably, a human pluripotent cell, more preferably a human pluripotent stem cell, or a cell differentiated from the cultured human pluripotent stem cell
  • a control sequence e.g., a promoter
  • a cell comprises the RNA genome according to any one of (18) to (24) above and/or the rhabdovirus according to (25) or (26) above.
  • FIG. 1A shows the RNA genomes of VSVs tested in the Examples.
  • the arrows indicate the position of the mutations made in the Example.
  • FIG. 1B shows the details of the mutations generated in the VSV.
  • FIG. 1C shows the schematic diagram for the experimental setup in Example 1.
  • FIG. 1D shows the results of the gene expression from the mouse ES cells infected with a VSV each containing the indicated RNA genome.
  • FIG. 1E shows the results of the gene expression from the mouse ES cells infected with a VSV each containing the indicated RNA genome.
  • the results contain the results from two independent experiments (i.e., Trial 1 and Trial 2).
  • a diagonal striped bar indicates the number of VSV positive cells
  • a black bar indicates the number of VSV negative cells
  • the number shown on each bar indicates the ratio of VSV positive cells to the total cells.
  • FIG. 2A shows the schematic diagram for the experimental setup in Example 2.
  • FIG. 2B shows the result of the gene expression from the mouse ES cells infected with a VSV under.
  • FIG. 3A shows the expression of a pluripotent marker OCT4 in the ESCs infected with the indicated 2mu-VSV or transfected with PiggyBac transposon vector.
  • FIG. 3B shows the expression of a pluripotent marker NANOG in the ESCs infected with the indicated 2mu-VSV or transfected with PiggyBac transposon vector.
  • FIG. 3C shows the expression of a pluripotent marker SSEA1 in the ESCs infected with the indicated 2mu-VSV or transfected with PiggyBac transposon vector.
  • FIG. 3D shows the schematic diagram for gene expression under the control of a guanine-responsive riboswitch from embryoid bodies (EBs) infected with the indicated 2mu-VSV or transfected with PiggyBac transposon vector.
  • EBs embryoid bodies
  • FIG. 3D shows the schematic diagram for gene expression under the control of a guanine-responsive riboswitch from embryoid bodies (EBs) infected with the indicated 2mu-VSV or transfected with PiggyBac transposon vector.
  • EBs embryoid bodies
  • FIG. 3E shows the gene expression from VSV in the absence of guanine and loss of the expression in the presence of guanine in a guanine-responsive riboswitch dependent manner.
  • FIG. 3F shows that the gene expression from the ESCs infected with the indicated 2mu-VSV disappeared at the outer periphery of each EB.
  • FIG. 3G shows that the gene expression of N protein from VSV also disappeared at the outer periphery of each EB.
  • FIG. 4 shows the number of cells treated with guanine.
  • FIG. 5A shows the structure of VSV-2mu vector and VSV-sc1 vector used in the experiments testing the cytotoxicity of these vectors.
  • FIG.5B shows the time course of the experiments testing the cytotoxicity of these vectors.
  • FIG. 5C shows the results showing the fluorescent microscope image of VSV-infected ES cells in the experiments testing the cytotoxicity of these vectors.
  • FIG. 5D shows the results showing the number of the total living ES cells. The number shown above each bar indicates the percentage of the fluorescent-positive ES cells to the total infected ES cells in each test group.
  • FIG. 6 shows the VSV-sc1 carrying MyoD gene for myogenic differentiation experiment in ES cells.
  • FIG.7 shows the time course of the myogenic differentiation experiment.
  • FIG. 8 shows the microscopic images of the ES cells infected with the VSV-sc1 carrying MyoD gene at the indicated time points.
  • FIG. 9 shows the images under the fluorescent microscopy showing the expression of myosin heavy chain (MHC) and VSV N protein (VSVN).
  • MHC myosin heavy chain
  • VSVN VSV N protein
  • FIG. 10 shows the expression of pluripotent markers (Oct 4 and Nanog) and skeletal muscle markers (Myogenin and Myf5) in the presence and absence of guanine.
  • the term “packaging” cell as used herein is a cell that produces a rhabdovirus or viral vector.
  • the viral genome of a virus or viral vector is engineered so that factors responsible for one or more functions selected from the group consisting of its multiplication, replication, and spread (including infection of other cells) are disrupted and the virus or viral vector cannot be multiplied, replicated, or spread after cell infection.
  • the virus may have been engineered so that it cannot multiply, replicate, or spread after cell infection.
  • packaging cells are supplemented with the destroyed factors so as to produce viral vectors to enable their multiplication, replication, and spread in the packaging cells.
  • packaging cells may express some of the factors that are destroyed in the virus to effectively produce a large amount of the rhabdoviruses or viral vectors.
  • Packaging cells may have such factors stably in their genome or transiently in an expression vector introduced into the packaging cells.
  • rhabdoviruses or rhabdoviral vectors have been classically obtained from a rhabdovirus or vector genome lacking gene encoding G protein (hereinafter also referred to as “G gene”).
  • Packaging cells may be supplemented with the G gene or foreign envelope protein to produce virions having a G protein or a foreign envelope protein on the surface of a virion of the rhabdovirus.
  • G gene-deleted viral genomes are preferably used in combination with packaging cells supplemented with G gene or G protein.
  • the DNA encoding a viral genome may be operably linked to a control sequence (e.g., promoter such as T7 promoter), and the production of the viral genome can be driven by the above control sequence.
  • a control sequence e.g., promoter such as T7 promoter
  • Such a DNA can be obtained by contacting the RNA genome of the rhabdovirus or rhabdoviral vector with a reverse transcriptase to obtain cDNA of the RNA genome. This allows the rhabdovirus genome to be produced from cDNA in packaging cells.
  • the T7 RNA polymerase can be supplied by a helper virus, such as vaccinia virus.
  • a packaging cell may be supplied with a gene encoding at least one or all of N, P, G, and L proteins operably linked to one or more control sequences that can drive transcription of the at least one or all of N, P, G, and L proteins, for example, by RNA polymerase (e.g., pol II), thereby supplying these virion components to form virions in the packaging cell.
  • RNA polymerase e.g., pol II
  • 293T cells have been used as a packaging cell.
  • the viral particles can be enriched, concentrated, and/or purified if needed before use.
  • a virus genome can contain an expression cassette comprising a control sequence and a foreign gene of interest operably linked to the control sequence.
  • control sequence is a sequence that has an activity to drive a gene operably linked thereto and to transcribe an RNA from the gene.
  • a control sequence is, for example, a promoter.
  • Promoters include, for example, class I promoters (which can be used for transcription of rRNA precursors), class II promoters (which contains a core promoter and an upstream promoter element and can be used for transcription of mRNA), and class III promoters (which are further classified into types I, II, and III).
  • the control sequence may also be referred to as a regulatory sequence and can be a promoter capable of transcribing mRNA in a cell such as an animal cell and a plant cell.
  • various pol II promoters can be used as a first control sequence.
  • pol II promoters include, but not limited to, CMV promoter, EF1 promoter (EF1 ⁇ promoter), SV40 promoter, MSCV promoter, hTERT promoter, ⁇ -actin promoter, CAG promoter, and CBh promoter.
  • Promoters can also include promoters that drive bacteriophage-derived RNA polymerases, such as the T7 promoter, T3 promoter, and SP6 promoter, as well as pol III promoters, such as the U6 promoter.
  • the T7 promoter is preferably used for transcription from a cyclic DNA
  • the SP6 promoter is preferably used for transcription from a linear DNA.
  • the promoter can also be an inducible promoter.
  • An inducible promoter is a promoter that can induce expression of a polynucleotide operably linked to the promoter only in the presence of an inducer that drives the promoter.
  • inducible promoter can induce expression of a polynucleotide operably linked to the promoter only in the absence of an inhibitor that suppresses the promoter activity.
  • Inducible promoters include, but not limited to, promoters that induce gene expression by heating, such as heat shock promoters.
  • Inducible promoters also include promoters that can be driven with a drug.
  • drug-inducible promoters include, for example, Cumate operator sequences, lambda operator sequences (e.g., 12 x lambda Op), and tetracycline-inducible promoters.
  • Tetracycline-inducible promoters include, for example, promoters that drive gene expression in the presence of tetracycline or its derivatives (e.g., doxycycline) or reverse tetracycline-regulated trans-activating factor (rtTA).
  • tetracycline-inducible promoter is the TRE3G promoter.
  • rhabdovirus refers to a virus belong to the family rhabdoviridae within the order Mononegavirales.
  • Rhabdoviruses have a non-segmented, negative-sense, single-stranded RNA genome having about 11 kb to about 16 kb in its length.
  • RNA genome commonly has a gene encoding N protein, a gene encoding P protein, a gene encoding M protein, a gene encoding G protein, and a gene encoding L protein from its 3’ end to 5’ end in this order.
  • the genes encoded are located from 3’ end to 5’ end in the genome.
  • RNA genome further has an accessory gene.
  • rhabdoviruses includes Lyssaviruses, Vesiculoviruses such as vesicular stomatitis virus (VSV) including vesicular stomatitis Indiana virus (VSIV), Ephemeroviruses, Novirhavdoviruses, and the other rhabdoviruses.
  • VSV vesicular stomatitis virus
  • VSIV vesicular stomatitis Indiana virus
  • Ephemeroviruses Ephemeroviruses
  • Novirhavdoviruses Ephemeroviruses
  • Novirhavdoviruses Novirhavdoviruses
  • the function of these viral proteins are as follows. N protein can bind to a viral RNA genome to form a nucleocapsid.
  • P protein can mediate the interaction between the nucleocapsid and L protein to facilitate the viral reproduction.
  • M protein can
  • PPxY sequence of M protein may be important for virus budding.
  • G protein is presented on the surface of a virion and is involved in binding to a receptor expressed on the surface of a cell and invasion into the cell.
  • a virion can be formed without G protein.
  • virion can present G protein or another foreign envelope protein to form a pseudo virus or a pseudo type virus.
  • L protein is an RNA-dependent RNA polymerase that can reproduce a viral RNA genome in infected cells.
  • L protein contains six domains I to IV, wherein domain III has a polymerase activity, and V and VI have an activity of capping mRNA.
  • N protein of the rhabdovirus may have an amino acid sequence that is orthologous or corresponding to an amino acid sequence set forth in GenBank Accession No.
  • P protein of the rhabdovirus may have an amino acid sequence that is orthologous or corresponding to an amino acid sequence set forth in GenBank Accession No. AAB60557.1.
  • M protein of the rhabdovirus may have an amino acid sequence that is orthologous or corresponding to an amino acid sequence set forth in SEQ ID No: 1 or GenBank Accession No. ACK43164.1.
  • G protein of the rhabdovirus may have an amino acid sequence that is orthologous or corresponding to an amino acid sequence set forth in GenBank Accession No. AAA48402.1.
  • L protein of the rhabdovirus may have an amino acid sequence that is orthologous or corresponding to an amino acid sequence set forth in SEQ ID No: 2 or GenBank Accession No.
  • ABP01784.1 These examples of the amino acid sequences are of vesicular stomatitis Indiana virus.
  • a VSV vector usually has N, P, M, and L proteins, and lacking G protein. This VSV can be made by deleting a gene encoding G protein from a VSV virus (e.g., a VSV or VSIV having an RNA genome that has a sequence corresponding to the sequence registered under GenBank accession number: MW373779.1).
  • a VSIV vector is a preferable embodiment of the VSV vector.
  • VSV vesicular stomatitis virus
  • VSIV vesicular stomatitis Indiana virus
  • G protein the envelope protein
  • VSVs can be engineered to be a virus that presents an envelope protein of another virus, which is known as a pseudo virus.
  • Such envelop proteins includes, for example, but not limited to, MHV-S of mouse hepatitis virus and GP64 of a virus in the family Baculoviridae.
  • VSVs can be an expression vector carrying an expression cassette expressibly containing an exogenous gene. VSVs may also be oncolytic.
  • an RNA genome of a rhabdovirus comprises at least a gene encoding M protein.
  • the gene encoding M protein preferably has mutations such that the gene cannot produce at least one of M2 and M3 isoforms, and preferably cannot produce both of M2 and M3 isoforms.
  • M2 isoform is known to have an amino acid sequence from position 33 and M3 isoform is known to have an amino acid sequence from position 51 in the full-length amino acid sequence set forth in SEQ ID No: 1 or GenBank Accession No. ACK43164.1.
  • the gene can be modified.
  • the gene expresses none of M2 and M3 isoforms by the deletion or substitution of methionine residues.
  • methionine can be substituted with another amino acid residue, for example, alanine or arginine at positions 33 and 51, preferably arginine at positions 33 and 51.
  • the RNA genome lacks gene encoding functional G protein. In an embodiment, the RNA genome may lack gene encoding G protein.
  • the RNA genome has a region comprising a gene encoding the M protein as mentioned above and a transcription end signal (TES) thereof.
  • a transcription end signal (TES) is located at the end of a gene encoding M protein.
  • the TES has a mutation selected from the group consisting of insertion, deletion, and substitution.
  • the TES has an insertion of an RNA sequence.
  • the RNA sequence may be an adenine ribonucleotide.
  • the TES has an insertion of an adenine ribonucleotide.
  • M1 to M3 isoforms can be produced. In an embodiment, only M1 can be produced, while no M2 and M3 isoforms can be produced.
  • the RNA genome has a gene encoding N protein.
  • the gene encoding N protein has a mutation, for example, in lysin (K) corresponding to the lysin positioned at 155 of N protein set forth in SEQ ID No. 16.
  • the N protein has a mutation in lysin that corresponds to isoleucine at position 155 of the amino acid sequence set forth in SEQ ID No: 16, wherein the mutation is a substitution with another amino acid, preferably with arginine (R).
  • the RNA genome has a gene encoding P protein.
  • the gene encoding P protein has a mutation, for example, in G corresponding to the G positioned at 645 of the nucleic acid sequence set forth in SEQ ID No. 17. This mutation is not considered to alter the amino acid sequence of P protein.
  • the RNA genome has a gene encoding L protein.
  • the gene encoding L protein has a mutation, for example, in domain III.
  • the gene encoding L protein has a mutation in isoleucine that corresponds to isoleucine at position 762 of the amino acid sequence set forth in SEQ ID No: 2 or GenBank Accession No. ABP01784.1.
  • the gene encoding L protein has a mutation in glutamine (Q) that corresponds to glutamine at position 1530 of the amino acid sequence set forth in SEQ ID No: 2 or GenBank Accession No. ABP01784.1.
  • the gene encoding L protein has a mutation in threonine (T) that corresponds to threonine at position 1910 of the amino acid sequence set forth in SEQ ID No: 2 or GenBank Accession No. ABP01784.1.
  • the gene encoding L protein has a mutation in g that corresponds to g at position 6204 of the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID No: 2 or GenBank Accession No. ABP01784.1.
  • the gene encoding L protein has one or more or all mutations in (i) isoleucine that corresponds to isoleucine at position 762 of the amino acid sequence set, (ii) glutamine (Q) that corresponds to glutamine at position 1530 of the amino acid sequence, and (iii) threonine (T) that corresponds to threonine at position 1910 of the amino acid sequence set forth, wherein the amino acid sequence is set forth in SEQ ID No: 2 or GenBank Accession No. ABP01784.1.
  • the gene encoding L protein has a mutation in (i) isoleucine (I) that corresponds to isoleucine at position 762 of the amino acid sequence; and one or more mutations in (ii) glutamine (Q) that corresponds to glutamine at position 1530 of the amino acid sequence, and (iii) threonine (T) that corresponds to threonine at position 1910 of the amino acid sequence set forth, wherein the amino acid sequence is set forth in SEQ ID No: 2 or GenBank Accession No. ABP01784.1.
  • the above-mentioned isoleucine (I) is substituted with leucine (L).
  • the above-mentioned glutamine (Q) is substituted with proline (P).
  • the above-mentioned threonine (T) is substituted with methionine (M).
  • the mutation in isoleucine is deletion or substitution with another amino acid such as leucine, preferably substitution with leucine.
  • M1 to M3 isoforms can be produced. In a preferable embodiment among these embodiments, only M1 can be produced, while no M2 and M3 isoforms can be produced.
  • the RNA genome has a region comprising a gene encoding the M protein as mentioned above and a transcription end signal (TES) thereof and a gene encoding L protein.
  • TES transcription end signal
  • the mutation in the TES and the mutation in L protein synergistically improve the viral replication and gene expression from the viruses.
  • the present disclosure provides the RNA genome has a region comprising a gene encoding the M protein as mentioned above and a transcription end signal (TES) thereof and a gene encoding L protein, wherein the TES has an insertion of an RNA sequence, and the gene encoding L protein has a mutation in isoleucine that corresponds to isoleucine at position 762 of the amino acid sequence set forth in SEQ ID No: 2 or GenBank Accession No. ABP01784.1.
  • TES transcription end signal
  • the RNA genome has a region comprising a gene encoding the M protein as mentioned above and a transcription end signal (TES) thereof and a gene encoding L protein, wherein the TES has an insertion of an adenine residue inside the TES and the L protein has a mutation in isoleucine that corresponds to isoleucine at position 762 of the amino acid sequence set forth in SEQ ID No: 2 or GenBank Accession No. ABP01784.1, wherein the mutation is substitution with another amino acid, preferably with leucine.
  • TES transcription end signal
  • a rhabdovirus may preferably be a vesicular stomatitis virus such as a vesicular stomatitis Indiana virus (VSIV).
  • VSIV vesicular stomatitis Indiana virus
  • the RNA genome may preferably contain a gene encoding N protein, a gene encoding P protein, a gene encoding the M protein, and a gene encoding the L protein.
  • the RNA genome may preferably lack a gene encoding functional G protein, preferably lack gene encoding G protein.
  • the RNA genome may contain an expression cassette containing a control sequence and a gene of interest such as an exogeneous gene operably linked to the control sequence.
  • the gene of interest can be expressed in a cell that the rhabdovirus has infected.
  • genes of interest include, but not limited to, one or more transcription factors selected from the group consisting of: DLX3 (distal-less homeobox 3), NEUROG3 (neurogenin 3), NEUROG2 (neurogenin 2), NEUROG1 (neurogenin 1), ASCL1 (achaete-scute family bHLH transcription factor 1), NEUROD1 (neurogenic differentiation 1), YY1 (YY1 transcription factor), SOX11 (SRY (sex determining region Y)-box 11), GLIS2 (GLIS family zinc finger 2), PDX1 (pancreatic and duodenal homeobox 1), E2F6 (E2F transcription factor 6), SOX2 (SRY (sex determining region Y)-box 2), CDX2 (caudal type homeobox 2), DLX4 (distal-less homeobox 4), NANOG (Nanoghomeobox), MXI1 (MAX interactor 1, dimerization protein), RNF2
  • SUZ12 SUZ12 polycomb repressive complex 2 subunit
  • JAG1 jagged 1
  • ATF3 activating transcription factor 3
  • ATF1 activating transcription factor 1
  • FLI1 Flu-1 proto-oncogene
  • ETS transcription factor ETS transcription factor
  • ETV5 ets variant 5
  • NELFA negative elongation factor complex member A
  • TCF23 transcription factor 23
  • ZNF646 zinc finger protein 646)
  • SIX5 SIX homeobox 5
  • MYBL2 v-myb avian myeloblastosis viral oncogene homolog-like 2
  • PAX6 paired box 6
  • SMAD2 SMAD family member 2
  • SOX9 ⁇ SRY SOX9 ⁇ SRY
  • STRA13 stimulated by retinoic acid 13
  • TBX6 T-box 6
  • SMAD1 SMAD1
  • one or more of Myod1, Mef2c, and Esx1 may be introduced into a pluripotent cell.
  • Myod1, Mef2c, and Esx1 may be introduced into a pluripotent cell.
  • Hnf4a, Foxa1, Gata2, and Gata3 may be introduced into a pluripotent cell.
  • TGIF, TCF4, PITX2, SALL4, and MEIS1 may be introduced into a pluripotent cell.
  • Sfpi1, Elf1, Elf5, Myc, Irf2, and Ets1 may be introduced into a pluripotent cell.
  • SPI1, OVOL2, CDX2, CEBPB, and SALL4 For differentiation into a nerve cell, one or more of Asc11 Smad7, Nr2f1, Sox11, Dmrt1, Sox9, Foxg1, and Sox2 may be introduced into a pluripotent cell.
  • NEUROD1, NEUROD2, NEUROG1, NEUROG2, and NEUROG3 may be introduced into a pluripotent cell.
  • NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB may be introduced into a pluripotent cell.
  • Other Examples of the gene of interest can be disclosed in US10836997B, which is herein incorporated by reference in its entirety.
  • a VSV vector according to the present disclosure that may carry said one or more of these genes may be introduced into the pluripotent cell to express said one or more of these genes in the pluripotent cell.
  • genes of interest include, but not limited to, a gene encoding a differentiation-inducing protein, a gene encoding a protein for a protein (e.g., enzyme) replacement therapy, a gene encoding a reprogramming factor, and a gene encoding a differentiation-suppressing protein.
  • a cell to be infected with the present virus may be a pluripotent cell or pluripotent stem cell, such as an embryonic stem cell (ES cell) and an induced pluripotent stem cell (iPS cell).
  • ES cell embryonic stem cell
  • iPS cell induced pluripotent stem cell
  • differentiation-inducing proteins may include activin A, bFGF, EGF, Esrrb, EBF1, C/EBP (e.g., C/EBP ⁇ , and C/EBP ⁇ ), Ngn3, Pdx, BMP, TGF ⁇ , and Mafa.
  • reprogramming factors may include Oct4, Sox2, Nanog, Klf such as Klf4, c-Myc, Nanog, Glis1, and Lin-28.
  • reprogramming factors are one or more or all selected from a group consisting of Oct4, Sox2, Klf, and c-Myc.
  • Example of a gene of interest include, but not limited to, one or more of a selection marker gene such as a drug-resistance gene, fluorescent gene, and an auxotrophic factor.
  • Examples of a gene of interest include, but not limited to, a nuclease involved in gene editing, such as zinc finger nuclease (ZFN), TALEN, and Cas nucleases of CRISPR/Cas system.
  • the RNA genome may contain an expression cassette containing a control sequence and a nucleic acid sequence encoding RNA such as siRNA, shRNA, miRNA, crRNA, tracrRNA, sgRNA, antisense RNA, and ncRNA, operably linked to the control sequence.
  • Expression cassette may express RNA such as siRNA, shRNA, miRNA, crRNA, tracrRNA, sgRNA, antisense RNA, and ncRNA.
  • RNA such as siRNA, shRNA, miRNA, crRNA, tracrRNA, sgRNA, antisense RNA, and ncRNA.
  • one or more of genes of interest is operably linked to an inducible regulator such as a riboswitch that is responsive to guanine, and thus, in the presence of guanine, the expression is suppressed, while, in the absence of guanine, the expression increases.
  • an inducible regulator such as a riboswitch that is responsive to guanine
  • the expression is suppressed, while, in the absence of guanine, the expression increases.
  • one or more of genes of interest is operably linked to an inducible promoter, and thus, the expression can be controlled by the addition of an inducer that can drive the inducible promoter.
  • a gene encoding L protein can be operably linked to an inducible regulator such as a guanine-responsive riboswitch.
  • a guanine-responsive riboswitch can suppress the expression of the gene under its control in the presence of a guanine molecule.
  • Such guanine-responsive riboswitch includes a guanine-responsive riboswitch as disclosed in Nomura et al., ACS Synthetic Biology, 2, 684-689 (2013), which is herein incorporated by reference in its entirety.
  • the inducible regulator will be inserted at 5’ untranslated region (5’UTR) just before a start codon, or preferably 3’ untranslated region (3’UTR) just before a poly-adenylation signal.
  • the term “untranslated region” (UTR) means a part of a transcript that is not translated into a protein.
  • a guanine-responsive riboswitch may be selected from the group consisting of Gua15HDV, Gua37HDV, GuaM0HDV, GuaM1HDV, GuaM7HDV, GuaM8HDV, GuaM9HDV, GuaM10HDV, GuaM11HDV, and GuaM12HDV (see Table 1).
  • a guanine-responsive riboswitch may be selected from the group consisting of Gua15HDV, Gua37HDV, GuaM1HDV, GuaM7HDV, GuaM8HDV, GuaM11HDV, and GuaM12HDV.
  • a guanine-responsive riboswitch may be GuaM8HDV.
  • a gene encoding a guanine-responsive riboswitch has sequences as shown in Table 1 instead of the sequence of the corresponding positions in positions 46 to 48 and 106 to 108 in SEQ ID No: 3.
  • a gene encoding a guanine-responsive riboswitch has the sequence set forth in SEQ ID No: 3.
  • a guanine-responsive riboswitch contains 1 st arm whose amino acid sequence is XUXXUA, a 1 st loop whose amino acid sequence is UAX, a 2 nd arm having 5 to 7 amino acids in length, a 2 nd loop having an amino acid sequence of XAUAXGG, a 3 rd arm that can hybridize with the 2 nd arm to form a stem, a 3 rd loop having an amino acid sequence of GUXUCUAC, a 4 th arm having an amino acid sequence of CXXXXXX, a 4 th loop having an amino acid sequence of CCXUAAA, a 5 th arm that can hybridize with the 4 th arm to form a stem, 5 th loop having an amino acid sequence of GAC, and a 6 th arm that can hybridize with the 1 st arm to form a stem, wherein these arms and loops are directly linked in this order, and wherein X represent A, U, G, or
  • Guanine can bind to A in the 1 st arm; U in the 1 st loop; the 1 st U, the 3 rd U, and A in the 3 rd loop, C in the 5 th loop, and U in the 6 th arm to change the conformation of the RNA to disrupt a following start codon, AUG (Mulhbacher et al., Plos Pathogens, 6(4): e1000865).
  • the RNA genome may comprise an expression cassette.
  • the gene is operably linked to a control sequence like a promoter such that the gene can be expressed in a cell of interest.
  • An expression cassette may comprise a control sequence, a translation initiation sequence such as an internal ribosome entry site (IRES), a start codon, a termination codon, and a transcription termination sequence.
  • a eukaryotic expression vector may comprise a Kozak consensus sequence.
  • the RNA genome may comprise an expression cassette between the genes encoding the viral components.
  • the RNA genome may lack the gene encoding G protein and may comprise an expression cassette between the gene encoding M protein and the gene encoding L protein.
  • a rhabdovirus comprising a virion having the RNA genome as explained above.
  • the virion may comprise N protein, P protein, the M protein, and L protein, with or without G protein or a foreign envelop protein.
  • the virion may comprise N protein, P protein, the M protein, L protein, and G protein or a foreign envelop protein.
  • At least one pluripotency marker genes may be maintained in the pluripotent cells infected with the rhabdovirus according to the disclosure.
  • Example of pluripotency marker genes include, but not limited to, Oct4, Nanog, Sox2, and SSEA1.
  • at least one pluripotency marker genes selected from the group consisting of Oct4, Nanog, and SSEA1 may be maintained in the pluripotent cells infected with the rhabdovirus according to the disclosure.
  • a composition comprising the rhabdovirus according to the invention.
  • the composition may be a pharmaceutical composition.
  • a pharmaceutical composition may comprise the rhabdovirus according to the invention and optionally a pharmaceutically acceptable excipient such as salts, pH buffer, osmotic agent, and solvent including water.
  • a pharmaceutical composition may be for use in treating a disease or condition that can be caused by a deficiency in a gene product such as an enzyme, comprising a rhabdovirus according to the disclosure comprising a gene encoding the deficient gene product.
  • a pharmaceutical composition may be used in an enzyme replacement therapy.
  • a pharmaceutical composition may be for use in treating cancer, due to an intrinsically oncolytic feature of a rhabdovirus.
  • a DNA encoding the RNA genome according to the invention can be produced by reacting a reverse transcriptase to the RNA genome under suitable conditions to obtain a complementary DNA (cDNA).
  • cDNA complementary DNA
  • a double-stranded DNA can be produced from cDNA with an appropriate primer and a DNA-dependent DNA polymerase under suitable conditions.
  • a double-stranded DNA can be incorporated into an expression vector that can express the RNA genome in a cell (i.e., an infected cell with the vector).
  • An expression vector may contain an origin of replication, a selectable marker such as a drug-resistance marker or a visible marker such as a fluorescent protein, and a suitable site for the insertion of a gene of interest, for example, a multiple cloning site.
  • a suitable site for the insertion of a gene of interest may be within an expression cassette.
  • the gene is operably linked to a control sequence like a promoter such that the gene can be expressed in a cell of interest.
  • Examples of cells to be infected includes, but not limited to, an animal cell, a human cell, and a mammalian cell.
  • a VSV can be produced in a 293 cell or 293T cell.
  • An expression cassette may comprise a control sequence, a translation initiation sequence such as a ribosomal binding site, a start codon, a termination codon, and a transcription termination sequence.
  • a eukaryotic expression vector may comprise a Kozak consensus sequence.
  • the virions produced in the cell can be collected as appropriate.
  • the intracellularly formed virions may be released extracellularly. Therefore, the virions can be recovered from the culture medium.
  • the recovered virions can be further isolated, enriched, purified, and/or concentrated. In this way, isolated, enriched, purified, or concentrated virus or viral vectors are provided.
  • the resulting viruses or viral vectors can be stored.
  • the resulting viruses or viral vectors can be stored, for example, in a deep freezer (e.g., at about -80°C), in a freezer (e.g., at about -20°C), or in a refrigerator (at about 4°C).
  • the resulting viruses or viral vectors can be stored in liquid nitrogen.
  • MOI Multiplicity of Infection
  • MOI may be optimized to achieve an effective infection to cells, and may be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more.
  • a cell comprising the RNA genome according to the invention and/or the rhabdovirus according to the invention.
  • a rhabdovirus may enter into the cell to obtain the cell comprising the rhabdovirus.
  • the RNA genome can be replicate in the cell.
  • the viral components carried in the RNA genome may be translated to form virions.
  • the RNA genome lacks a gene encoding G protein.
  • examples of cell include, but not limited to, a eukaryotic cell such as an animal cell and a plant cell.
  • An animal cell can be a mammalian cell, for example, a human cell.
  • a human cell includes, for example, but not limited to a pluripotent cell such as a pluripotent stem cell (e.g., embryonic stem cell (ES cell) and induced pluripotent stem cell (iPS cell)), a multipotent cell, a stem cell, a tissue progenitor cell, and a somatic cell.
  • a pluripotent stem cell e.g., embryonic stem cell (ES cell) and induced pluripotent stem cell (iPS cell)
  • ES cell embryonic stem cell
  • iPS cell induced pluripotent stem cell
  • a VSV according to the disclosure can maintain the pluripotency of infected pluripotent cells, and further that a VSV can disappear after the infected pluripotent cells differentiate into a next developmental stage. It has been known that a VSV is an RNA virus and thus can produce no integration into the host genome and no damage on the host genome. The feature of a VSV capable of disappearing after the infected pluripotent cells differentiate into a next developmental stage will be suitable for culturing a pluripotent cell and inducing differentiation of the pluripotent cell.
  • a composition comprising a cell according to the disclosure may be provided.
  • a pluripotent cell that has been infected with a VSV according to the present disclosure can be maintained in a culture medium under the condition suitable for culturing the pluripotent cell.
  • a culture medium for the pluripotent cell may be a serum-free medium or a medium containing serum.
  • a culture medium for the pluripotent cell contains a MEK inhibitor such as PD0325901, ROCK inhibitor such as Y-27632, TGF ⁇ inhibitor such as SB431542.
  • BRC6 mouse ESCs were obtained from RIKEN BioResource Center (Tsukuba, Japan). Guanine (G11950-10G, Sigma-Aldrich) was dissolved in 0.2 N NaOH at 20 mM as a stock solution and was used at the final concentration of 100 ⁇ M.
  • BRC6 cells were cultured in the following medium (ESM): Glasgow’s Minimum Essential Medium (11710-035, Gibco, Themo Fisher Scientific) containing 15% (v/v) KnockOut Serum Replacement (10828-028, Gibco), 0.3% (v/v) fetal bovine serum (FBS) (10437-028, Gibco), 2 mM L-glutamine (25030-081, Gibco), 1 mM sodium pyruvate (11360-088, Gibco), 1 ⁇ MEM Non-Essential Amino Acids Solution, (11140-050, Gibco), 0.1 mM 2-mercaptoethanol (21985-023, Gibco), 1 ⁇ Penicillin-Streptomycin (15140-122, Gibco), 1 ⁇ M PD0325901 (04-0006, Stemgent, REPROCELL), 3 ⁇ M CHIR99021 (SML1046-5MG, Sigma-Aldrich), and 1000 U/m
  • BHK21 and 293T cells (ECACC: 12022001, DS Pharma Biomedical) were maintained on 10 cm dishes (3020-100, IWAKI) in Dulbecco's modified Eagle's medium (high glucose) (044-29765, Wako) supplemented with 10% (v/v) FBS, 1 ⁇ Penicillin-Streptomycin, and 2 mM L-alanyl-L-glutamine (016-21841, Wako). The cells were passaged using TrypLE Express every 3-4 days. Cultured cells were observed with an inverted microscope (ECLIPSE Ts2, Nikon) and photographed using a digital camera (D7500, Nikon).
  • Plasmids carrying the VSV genomes were constructed based on VSV ⁇ G(GFP)_GFP/L-GuaM8( Takahashi & Yokobayashi, ACS Synthetic Biology, 8, 1976-1982 (2019)) which contains a GuaM8HDV riboswitch in the 3’ UTR of EGFP and L protein genes, following the plasmid construction strategy described in our previous report (see Takahashi & Yokobayashi, ACS Synthetic Biology, 8, 1976-1982 (2019).).
  • VSV vector plasmids were transformed in NEB Stable Competent E. coli (C30401, New England Biolabs) and incubated 30°C.
  • the plasmids were purified from the cells using Zyppy Plasmid Miniprep Kit (D4037, Zymo Research) for Sanger sequencing and using QIAGEN Plasmid Maxi Kit (12163, QIAGEN) for transfection into 293T cells for virus production.
  • VSV particles were prepared as previously described (Takahashi and Yokobayashi, 2019). Briefly, 293T cells (1 ⁇ 10 6 ) were seeded in one of the wells in a 6-well plate and incubated at 37°C at 5% CO 2 overnight.
  • VSV vector plasmid (5 ⁇ g) was mixed with the following helper plasmids that express VSV proteins and T7 RNA polymerase: 1.5 ⁇ g pCAG-VSVN (Addgene: #64087), 2.5 ⁇ g pCAG-VSVP (Addgene: #64088), 0.5 ⁇ g pCAG-VSVL (Addgene: #64085), 4 ⁇ g pCAG-VSVG (Addgene; #35616), and 5 ⁇ g pCAG-T7 RNA polymerase (Addgene; #59926).
  • helper plasmids that express VSV proteins and T7 RNA polymerase: 1.5 ⁇ g pCAG-VSVN (Addgene: #64087), 2.5 ⁇ g pCAG-VSVP (Addgene: #64088), 0.5 ⁇ g pCAG-VSVL (Addgene: #64085), 4 ⁇ g pCAG-VSVG (Addgene; #35616),
  • the plasmids were mixed with 37 ⁇ L of TransIT-293 (MIR2700, Mirus Bio) and 250 ⁇ L OPTI-MEM (31985062, Gibco) and incubated at room temperature for 20 min, and then added to 293T cells and incubated for 4 h. After transfection, the medium was replaced with fresh medium and cultured for several days until red fluorescent cells appeared. The supernatant containing the viral particles was harvested and filtered with a syringe filter (0.22 ⁇ m) (2-856-01, AS ONE) which was used to infect BHK21 cells for viral amplification directly or after concentration using PEG-it solution (LV810A-1, System Biosciences).
  • BHK21 cells (2 ⁇ 10 5 ) were seeded in one of the wells in a 6-well plate and cultured overnight. The next day, the cells were transfected with 2 ⁇ g of pCAG-VSVG with 6 ⁇ L Fugene HD (E2311, Promega) in 150 ⁇ L OPTI-MEM after 5 min incubation at room temperature, and the cells cultured for one more day. The supernatant containing the viral particles from 293T transfection was added to amplify the viral particles. The amplification in BHK21 cells was repeated one more time, and the harvested viral particles were concentrated by PEG-it solution.
  • Fugene HD E2311, Promega
  • the viral particles were diluted in PBS (D-PBS(-), 045-29795, Wako) and divided into several aliquots to be stored at -80°C until use.
  • PBS D-PBS(-), 045-29795, Wako
  • the viral titers were determined as follows.
  • the viral stocks used in each experiment (WT, mM, +a, mL, 2mu) were thawed at the same time and their titers were measured on BHK21 cells.
  • Each viral aliquot was diluted with PBS by 1 ⁇ 10 4 -, 1 ⁇ 10 5 -, 1 ⁇ 10 6 -, and 1 ⁇ 10 7 -fold.
  • the diluted viral stocks (10 ⁇ L) were added to BHK21 cells in a 12 well plate containing 1 ⁇ 10 5 cells/well seeded the day before. Red fluorescent cells were counted using an inverted microscope after 2 days of culture. The viral aliquots thawed for titer measurements were immediately frozen at -80°C and used for experiments within 1 week after confirming the titers.
  • Mouse ESCs were dissociated by TrypLE Express and incubated on a gelatin-coated 6 well plate for 1 h at 37°C to remove the feeder cells as much as possible. Then, the supernatant containing the mouse ESCs was collected in a 15 mL tube and the cells were counted using Trypan Blue solution. An appropriate number of mouse ESCs were suspended in 1 mL ESM (without small molecule supplements), viral particles, and 10 ⁇ g/mL polybrene (12996-81, Nacalai Tesque) in a 1.5 mL microtube.
  • the cells were then incubated at room temperature while rotating for 1 h, centrifuged at 200 g, washed twice with 1 mL ESM, and suspended in an appropriate medium for the experiments.
  • the infected cells were passaged on gelatin-coated ⁇ -Slide 8 well (ib80826, ibidi, Martinsried, Germany) at 2 ⁇ 10 4 cells/well and cultured for 3 days in ESM.
  • Embryoid bodies were formed by the hanging drop method in the following medium (EBM) (Ohnuki and Kurosawa, 2013): Iscove's Modified Dulbecco's Medium containing 10% (v/v) FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 ⁇ MEM non-essential amino acids, 0.1 mM 2-mercaptoethanol, and 1 ⁇ Penicillin-Streptomycin.
  • Infected or uninfected mouse ESCs were suspended in 30 ⁇ L EBM droplets containing 1000 cells per droplet on the cover of a sterile petri dish (BME-SNS0001, BMBio). The covers were then inverted to prepare hanging droplets containing mouse ESCs.
  • the cells were cultured at 37°C in 5% CO 2 for 3 days in the droplets. After imaging, the formed EBs were transferred to the gelatin-coated ⁇ -Slide 8 well and cultured for 3 days in EBM for immunofluorescence staining.
  • the cells were fixed with 4% paraformaldehyde (50-980-495, Electron Microscopy Sciences) in PBS for 10 min at room temperature. The cells were then incubated in PBS containing 5% goat serum (31872, Invitrogen) and 0.1% Triton X-100 (T8787, Sigma-Aldrich) (without Triton X-100 for SSEA1) for 1 h at room temperature. The primary antibodies were then added to each well and incubated at 4°C overnight.
  • the primary antibodies were diluted in PBS containing 1% goat serum as follows: Oct-3/4 (C-10) (sc-5279, Santa Cruz Biotechnology), 1:50; Anti-Nanog (ab80892, abcam), 1:300; Alexa488-conjugated SSEA-1 (53-8813-42, eBioscience), 1:50; Anti-VSVN (10G4) (EB0009, Kerafast).
  • the secondary antibodies were added to each well and incubated for 45 min at room temperature after washing with PBS three times (10 min each).
  • the secondary antibodies were diluted in PBS as follows: Alexa Fluor 488 goat anti-mouse IgG (H+L) (A11001, Invitrogen) for Oct-3/4 and VSVN, 1:500; Alexa Fluor 488 goat anti-rabbit IgG (H+L) (A11008, Invitrogen) for Nanog, 1:500.
  • Alexa Fluor 488 goat anti-mouse IgG (H+L) A11001, Invitrogen
  • Alexa Fluor 488 goat anti-rabbit IgG H+L
  • 1 ⁇ g/mL of Hoechst 33342 (H1399, Invitrogen) in PBS was then added to each well and incubated for 5 min at room temperature.
  • Uninfected and infected mouse ESCs were dissociated by TrypLE Express and suspended in PBS for flow cytometry. The cells were analyzed on a flow cytometer (BD FACSAria III, BD). DsRedExpress2 expression was detected with the DsRed channel. As negative and positive controls, uninfected mouse ESCs and CAG-driven DsRedExpress2-expressing mouse ESCs, respectively, were used in each experiment. The data acquired from Aria III were analyzed by FlowJo (BD).
  • Cytotoxicity of VSV is a critical obstacle for applications of VSV as a transgene vector in vertebrate cells.
  • M protein matrix
  • the M protein is a dominant factor that induces an antiviral response in the infected cells via the type 1 interferon pathway, eventually leading to cell death. It has been reported that the gene encoding the M protein yields truncated translation products initiating at internal methionine residues M33 and M51. Suppression of these alternative translation products by mutating these methionines reduced cytopathogenicity. Consequently, the inventors introduced mutations M33R and M51R in the M protein in WT-VSV to yield mM-VSV (FIG. 1A).
  • the two strains each contained 3 and 5 mutations relative to the parental mM-VSV, but they shared two common mutations: insertion of an additional adenine in the transcription end signal (TES), whose original sequence is set forth in SEQ ID No: 4 (tatgaaaaa) of the M protein-coding region to form a mutated nucleic acid sequence set forth in SEQ ID No.: 5 (tatgaaaaaa) (hereinafter referred to as “+a-VSV”), and a point mutation in the coding region of the L protein (hereinafter referred to as “mL-VSV”) which results in the substitution of I762 to leucine (FIG. 1A).
  • a mouse ESC clone stably expressing DsRedExpress2 from the CAG promoter was generated using a PiggyBac transposon vector (FIGs. 1D, 3A-3C, 3E-3G).
  • +a-VSV and mL-VSV increase the total number of cells as well as the number of red fluorescent cells, compared with WT-VSV, and further, 2mu-VSV topped the total number of cells as well as the number of red fluorescent cells on day 1.
  • VSV vectors Chemical regulation of viral replication and transgene expression in mouse ESCs
  • the constructed VSV vectors are equipped with guanine responsive riboswitches (GuaM8HDV; SEQ ID No.: 3) that downregulate mRNA levels of a transgene encoding DsRedExpress2 and the L protein-coding gene in response to guanine (FIG. 1A).
  • guanine responsive riboswitches (GuaM8HDV; SEQ ID No.: 3) that downregulate mRNA levels of a transgene encoding DsRedExpress2 and the L protein-coding gene in response to guanine (FIG. 1A).
  • Mouse ESCs infected with 2mu-VSV maintain pluripotency
  • Mouse ESCs have the potency to differentiate into any cells of the whole body, including germ cells.
  • the characteristics of mouse ESCs are called naive state, while those of human ESCs are primed state, having more limited differentiation ability.
  • One of the hallmarks of naive pluripotency of mouse ESCs is their characteristic dome-shaped and compact colonies, while primed ESCs form flat colonies.
  • 2mu-infected cells formed colonies with somewhat looser cell-cell contacts while maintaining some mouse ESC-like 3D colony shape. (FIGs. 1D, and 2B). Therefore, the inventors decided to evaluate the pluripotency of the 2mu-VSV-infected mouse ESCs.
  • pluripotency markers To confirm the expression of pluripotency markers, immunofluorescence staining of pluripotency-related proteins was performed with uninfected and 2mu-VSV-infected mouse ESCs. OCT4 and NANOG, the hallmarks of pluripotent stem cells, were expressed in both uninfected and infected cells (FIGs. 3A and 3B). SSEA1, a mouse ESC marker, was observed on the cell surface (FIG. 3C). High alkaline phosphatase activities were also detected in both uninfected and infected mouse ESCs. (FIG. 3C).
  • mice infected with 2mu-VSV and uninfected cells were suspended in medium without LIF using the hanging drop method (FIG. 3D) to form spherical embryoid bodies (EBs) (FIGs. 3D, and 3E).
  • EBs spherical embryoid bodies
  • Three days of culture in the presence and absence of guanine (100 ⁇ M) confirmed chemical regulation of transgene expression during differentiation (FIG. 3E).
  • the EBs were transferred to a flat gelatin-coated dish (FIG. 3D). The differentiated cells attached and radially propagated on the bottom of the gelatin-coated dish (FIGs.
  • mouse ESCs infected with 2mu-VSV express pluripotency markers and can be differentiated into EBs in the differentiation induction medium.
  • 2mu-VSV replication appears to be attenuated as the differentiation progresses possibly due to maturation of the innate antiviral response.
  • VSV-sc1 A new VSV mutant clone isolated from VSV-infected mouse ES cells A further VSV mutant clone was isolated from the VSV-infected mouse ES cells. This newly isolated VSV mutant clone was named as VSV-sc1. VSV-sc1 was stably replicated in mouse ESCs. The genome sequence analysis of the VSV-sc1 reveals that the VSV-sc1 has further five new mutations in the VSV genome in addition to the original mutations in +a-VSV and mL-VSV.
  • the VSV-sc1 has 464A>G in a gene encoding N protein, 645G>A in a gene encoding P protein, 2284A>C, 4589A>C, 5729C>T, and 6204G>A in a gene encoding L protein, compared to the wild type VSV genome sequence registered as NCBI Reference Sequence NC_001560.1 (see Fig. 5A).
  • DsRedEx2 encoding a fluorescent protein was integrated into 3’ side of the gene encoding N protein in VSV-2mu and VSV-sc1, respectively, under the regulation of a guanine riboswitch.
  • VSV-sc1 MyoD and guanine riboswitches were inserted into the VSV-sc1 genome as depicted in Fig. 6, such that the expression of MyoD can be controlled by guanine. Further, Puromycin resistance gene (puromycin N-acetyl transferase) was inserted to select vector-infected stem cells. The obtained VSV-sc1 was referred to as VSV-MyoD.
  • Myogenic differentiation was carried out as shown in Fig. 7.
  • Mouse ESCs were infected with VSV-MyoD and cultured in the presence of 10 ⁇ g/mL puromycin and 100 ⁇ M guanine, and passaged nine times.
  • the infected ESCs were cultured in medium (containing GKF, LIF, and 2i) suitable for maintenance of pluripotency of ESCs for one day.
  • the guanine was removed from the medium by replacing the medium with fresh medium without guanine and was cultured for one day.
  • the medium was replaced with fresh DMEM medium supplemented with B27 for seven days.
  • the treated cells were observed by microscopy to obtain images of the treated cells. The obtained images are shown in Fig. 8.
  • the treated cells in the absence of guanine formed spindle-like cells, which were emerged at day 5 after being placed under the guanine free conditions., which the treated cells in the presence of guanine (Guanine +) formed no such spindle-like cells.
  • the treated cells were subjected to immunofluorescent staining by using anti-myosin heavy chain (MHC) antibody and anti-VSVN antibody as a primary antibody and Alexa-488-labeled mouse IgG as a secondary antibody, detecting and were observed by fluorescent microscopy.
  • MHC myosin heavy chain
  • MHC has been observed only in the cells treated in the absence of guanine, which confirms that the engineered VSV is useful as an expression vector for animal cells, and is suitable for the differentiation of treated cells.
  • mRNAs’ expressions were measured by quantitative-PCR, and the expression level was normalized as a relative expression against GAPDH mRNA expression (see Fig. 10), which is consistent with the results shown in Figs. 7 and 9.
  • SEQ ID No: 1 Amino acid sequence of M protein of VSIV
  • SEQ ID No. 2 Amino acid sequence of L protein of VSIV
  • SEQ ID No. 3 Nucleic acid sequence of a guanine-responsive riboswitch, GuaM8HDV
  • SEQ ID No. 4 Nucleic acid sequence of the wildtype part in the TES of M protein
  • SEQ ID No. 5 Nucleic acid sequence of the mutated part in +a-VSV
  • SEQ ID No. 6 Nucleic acid sequence of a part of wildtype M protein
  • SEQ ID No. 7 Amino acid sequence of a part of wildtype M protein SEQ ID No.
  • Nucleic acid sequence of a part of the mutated M protein SEQ ID No. 9 Nucleic acid sequence of a part of the mutated M protein SEQ ID No. 10-: Nucleic acid sequence of a part of wildtype 3’UTR of a gene encoding M protein SEQ ID No. 11: Nucleic acid sequence of a part of the mutated 3’UTR of a gene encoding M protein SEQ ID No. 12: Nucleic acid sequence of a part of wildtype L protein SEQ ID No. 13: Amino acid sequence of a part of wildtype L protein SEQ ID No. 14: Nucleic acid sequence of a part of the mutated L protein SEQ ID No. 15: Amino acid sequence of a part of the mutated L protein SEQ ID No. 16: Amino acid sequence of N protein of VSIVSEQ ID No. 17: Nucleic acid sequence of wildtype P protein

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Abstract

L'invention concerne un rhabdovirus modifié qui peut présenter une toxicité réduite et/ou induire une capacité de réplication plus élevée et/ou exprimer un gène chargé sur le virus dans une cellule infectée.
PCT/JP2022/019633 2021-05-10 2022-05-09 Rhabdovirus modifié ayant une toxicité réduite WO2022239724A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005098009A2 (fr) * 2004-04-09 2005-10-20 Wyeth Affaiblissement par effet de synergie du virus de la stomatite vesiculaire (vsv), vecteurs correspondants, et compositions immunogenes correspondantes
US20100172877A1 (en) * 2009-01-08 2010-07-08 Yale University Compositions and methods of use of an oncolytic vesicular stomatitis virus
WO2011056993A1 (fr) * 2009-11-04 2011-05-12 Yale University Compositions et procédés destinés à traiter le cancer par des virus oncolytiques atténués
US10836997B2 (en) 2015-03-09 2020-11-17 Keio University Method for differentiating pluripotent stem cells into desired cell type

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005098009A2 (fr) * 2004-04-09 2005-10-20 Wyeth Affaiblissement par effet de synergie du virus de la stomatite vesiculaire (vsv), vecteurs correspondants, et compositions immunogenes correspondantes
US20100172877A1 (en) * 2009-01-08 2010-07-08 Yale University Compositions and methods of use of an oncolytic vesicular stomatitis virus
WO2011056993A1 (fr) * 2009-11-04 2011-05-12 Yale University Compositions et procédés destinés à traiter le cancer par des virus oncolytiques atténués
US10836997B2 (en) 2015-03-09 2020-11-17 Keio University Method for differentiating pluripotent stem cells into desired cell type

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. MW373779.1
"NCBI", Database accession no. NC_001560.1
KIM NARAE ET AL: "Novel RNA Viral Vectors for Chemically Regulated Gene Expression in Embryonic Stem Cells", ACS SYNTHETIC BIOLOGY, vol. 10, no. 11, 22 October 2021 (2021-10-22), Washington DC ,USA, pages 2959 - 2967, XP055948468, ISSN: 2161-5063, Retrieved from the Internet <URL:https://pubs.acs.org/doi/pdf/10.1021/acssynbio.1c00214> DOI: 10.1021/acssynbio.1c00214 *
MULHBACHER ET AL., PLOS PATHOGENS, vol. 6, no. 4, pages el000865
NOMURA ET AL., ACS SYNTHETIC BIOLOGY, vol. 2, 2013, pages 684 - 689
TAKAHASHIYOKOBAYASHI, ACS SYNTHETIC BIOLOGY, vol. 8, 2019, pages 1976 - 1982

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